Use of apotransferrin that has bound zinc or copper ions for treatment of toxic effects of endotoxins

ABSTRACT

Apotransferrin bound to zinc or copper is administered to a patient who has endotoxins present in the patient&#39;s bloodstream. The apotransferrin improves the elimination of endotoxins from the bloodstream.

BACKGROUND OF THE INVENTION

The invention relates to the use of apotransferrin that has bounddivalent zinc or copper ions as an agent for prophylactic andtherapeutic treatment of the toxic effects of endotoxins. Apotransferrinis a glycoprotein with an average molecular weight of 80,000 Dalton. Itcan bind mainly trivalent iron ions reversibly. It can also bind othermetal ions instead of iron such as Cu(II), Mn(III), Co(III) or Zn(II).In human and animal organisms, apotransferrin usually occurs astransferrin, a protein-metal complex with trivalent iron.

The main function of apotransferrin is considered to be its ability tobind and transport specific trivalent iron ions, whereby it can bind andtransport one or two molecules of iron reversibly. In this way, the ironis transported following its absorption in the small intestine to theiron depots in the liver and spleen as well as in the reticulocytes inthe hematopoietic tissue. The normal range of plasma apotransferrin-ironcomplex (transferrin) concentration is 200 mg/dl to 400 mg/dl.

A further important function of transferrin in its iron-free form isconsidered to be its bacteriostatic effect (Martin CM, Jandl JH, FinlandM. J Infect Dis 1963; 112:158-163; Fletcher J. Immunology 1971;20:493-500). Iron is an essential growth factor for bacteria. Thecomplexing of iron by transferrin keeps the free iron concentration inthe plasma under the minimum required for bacterial growth. Berger undBeger (Berger D, Beger HG. Clin Chim Acta 1987; 163:289-299;Arzneim-Forsch/Drug Res 1988; 38:817-820; and Prog Clin Biol Res 1988;272:115-124) concluded on the basis of the bacteriostatic effect oftransferrin that transferrin in its iron-free form might even fulfil thefunction of an adjunctive anti-microbial agent in extensivegram-negative infections in the sense of a complement to conventionalantibiotic therapy.

Apotransferrin that has bound trivalent iron, i.e. in its form astransferrin, is also capable of reducing the biological activity ofendotoxins. The ability of transferrin to neutralize endotoxin increasesalong with the iron load as is described in detail in DE 38 42 143 andDE 38 44 667. The fact that apotransferrin, having bound iron ions, ismore or less capable of neutralizing endotoxins, depending on the ironlevel, does not necessarily mean that the same effect will achieved whenother metal ions are bound.

In contrast to the conclusions described in DE 38 42 143 and in DE 3844667, Berger et al. report that ". . . the endotoxin binding capacity isrestricted to apotransferrin." (Berger D, Winter M, Beger HG. Clin ChimActa 1990; 189:1 (Summary)) and that the ability of apotransferrin tobind and neutralize endotoxin is reduced when iron is bound (Berger D,Beger HG. Clin Chim Acta 1987; 163:289-299; Arzneim-Forsch/Drug Res1988; 38:817-820; and Prog Clin Biol Res 1988; 272: 115-124; LangenbecksArchiv fur Chirurgie 1990; Suppl. 1-6; Berger D, Winter M, Beger HG.Clin Chim Acta 1990, 189: p. 1-6). The authors even mention that"transferrin . . . exhibits endotoxicity enhancing activity." (Berger D,Winter M, Beger HG. Clin Chim Acta 1990; 189: p. 4). Berger et al. alsoreport that, besides pH, the presence of divalent cations (Ca²⁺, Mg²⁺,Mn²⁺) in the reaction medium is of decisive importance regarding thebinding of endotoxin by apotransferrin (Berger D, Beger HG.Arzneim-Forsch/Drug Res 1988; 38:817-820; Prog Clin Biol Res 1988; 272:115-124; Berger D, Winter M, Beger HG. Clin Chim Acta 1990, 189: p. 1-6and Berger D, Kitterer WR, Beger HG. Eur J Clin Invest 1990; 20:66-71).The authors merely mention that divalent cations must be present in thereaction mixture at a concentration of 2 mmol/l or 3 mmol/l (Mg²⁺) anddo not describe a potential influence of ion concentration on thebinding of endotoxin. The authors do not conclude that an increase inthe ion concentration in the solution, and an indirectly relatedincrease in apotransferrin-cation complexes, result in an improvement ofendotoxin binding by apotransferrin. Thus these publications contain noindication whatever that a primary binding of cations to apotransferrincould be one of the preconditions for the binding of endotoxins toapotransferrin.

Endotoxin is a constituent of the cellular walls of gram-negativebacteria and is released only by the bacterial decay. It is amacromolecule with a molecular weight of up to 1×10⁶ Dalton, consistingmainly of sugar compounds and fatty acids. It may also include complexedprotein residues from the wall of the bacteria. The endotoxin moleculeconsists of three structurally and immunobiologically differentsubregions:

Subregion 1, the O-specific chain, consists of several repetitiveoligosaccharide units, each of which is made up of a maximum of 5neutral sugars. The number of oligosaccharides present depends on thestrain of bacteria; for example, the endotoxin of S. abortus equi usedin our experiments has 8 oligosaccharides in this region.

Subregion 2, the core oligosaccharide, consists, among other things, ofn-acetyl-glucosamine, glucose, galactose, heptose and2-keto-3-desoxyoctone acid.

Subregion 3, The lipid A (MW 2,000 Dalton) consists of a phosphorylatedD-glucosamine-disaccharide to which several--approx. 7-long-chain fattyacids are bound as amides and esters. The carrier of the toxicproperties is the lipid A, whereby the toxic effects derive from severalfatty acid residues in this region.

The size of the endotoxin molecule and its charge characteristics allowfor complexing various compounds and proteins with the groups orside-chains of the three subregions in the endotoxin structure withoutthis having any influence on its toxic properties. Normally, there is aprotein bound to the lipid A, the so-called lipid A-associated protein.In most cases, separation of this protein component from the lipid Acauses no change whatever in the toxic effect in some endotoxins. Itwas, however, found that binding of proteins onto many endotoxins canalso result in a considerable increase in their toxicity (RietschelE.TH. et al. (1982)): "Bacterial Endotoxins: Chemical Structure,Biological Activity and Role in Septicaemia", Scand. J. Infect. Dis.Suppl. 31: 8-21). For example, it was observed that certain endotoxinsthat have complexed a certain amount of protein can be 100 times astoxic as endotoxins from which the same protein was separated (MorrisonDC, Oudes ZG, Betz J (1980): The role of lipid A and lipid A-associatedprotein in cell degranulation mechanisms. In: Eaker D. Wadstrom T (Eds).NATURAL TOXINS, pp 287-294, Pergamon Press. New York). In animalexperiments as well, a decrease in toxic effect was observed when theprotein was split off (Hitchcock PJ, Morrison DC. (1984). "The proteincomponent of bacterial endotoxins". In: E.T. Rietschel (editor) HANDBOOKof ENDOTOXINS, Vol 1: Chemistry of Endotoxin, pp 339-375, ElsevierScience Publishers B.V.).

Free, i.e. biologically active, endotoxin cannot normally be detected inthe blood of healthy subjects. In the following pathological conditions,however, increased amounts of biologically active endotoxin may occur inthe blood:

a) Increased transfer of endotoxin from the intestinal tract into theblood on account of permeability disturbances in the intestinal wall,e.g. in severe enterocolitis, shock, or increased release in the courseof enteral antibiotic therapy.

b) Reduced eliminetion of endotoxins by the liver, e.g. liverdysfunction partial liver resection.

c) Increased endotoxin release from a larger gram-negative focus such ascould be found in treatment of peritonitis with antibiotics.

To protect organs from damage, plasma in healthy persons can inactivatethe endotoxin that is continually transferred from the intestinal tract.An excessive endotoxin transfer into the blood brings about a rapidexhaustion of endotoxin-inactivating capacity of the plasma, so thateven more biologically active endotoxin is found in the plasma, leadingto the clinical signs of endotoxemia. If this condition continues,endotoxemia may lead to cell decay, and finally to organ failure. Forthese reasons, additional therapeutic measures are required to reduceblood endotoxin activity whenever increased endotoxin passage into theblood or reduced hepatic endotoxin elimination are to be expected.

To a certain extent, plasma endotoxin activity can be reduced byadministering polyvalent 7S-IgG preparations, presumably due to thelipid A antibodies they contain. A further improvement in endotoxinneutralization is achieved by enriching the IgG preparation withimmunoglobulin of the IgM fraction, in which a higher level of lipid Aantibody titer is present (Appelmelk BJ et al., Microbiol Pathogenesis1987; 2: 391-393).

Clinical studies (Baumgartner JD, et al. Prevention of gram-negativeshock and death in surgical patients by antibody to endotoxin coreglycolipid. Lancet 1985;ii: 59-63; Dunn DL, Priest BP, Condie RM.Protective capacity of polyclonal and monoclonal antibodies directedagainst endotoxin during experimental sepsis. Arch Surg 1988;123:1389-1393; Ziegler EJ. et al. Treatment of gram-negative bacteremiaand septic shock with HA-1A human monoclonal antibody against endotoxin.N Engl J Med 1991, 324: 429-436) have demonstrated that lethality ofsepticemia can be reduced by decreasing plasma endotoxin activity ifmonoclonal antibodies to the core or the lipid A portion of theendotoxin are administered.

An alternative to the neutralization of endotoxin by monoclonalantibodies is neutralization of the toxic effect of endotoxin bytransferrin (DE 38 44 667). The combination of transferrin withpolyvalent immunoglobulin preparations can achieve a synergistic effectas described in DE 38 42 143.

As described in E 38 44 667 in detail, the capacity of apotransferrin toneutralize endotoxin increases as the content of trivalent iron ionsincreases. In the organism, however, the iron can be split off from thetransferrin by means of a reduction to Fe²⁺. Since the oxygen activationof enzyme-coupled reactions, i.e. formation of oxygen radicals byneutrophil granulocytes, can be stimulated by an increased presence ofFe²⁺ ions, it is possible that transferrin therapy may facilitate theundesirable formation of harmful oxygen radicals in the organism.However, non-enzymatic processes that lead to oxygen activation and thedamaging oxygen radicals it entails, such as the so-called Haber-Weissreaction (Haber F., Weiss J. Proc Royal Soc [A]1934; 147: 332-351) maybe catalysed by the reduced divalent iron split off from transferrin(Carlin G., Djursater R. FEBS Lett 1984; 177: 27-30). The raised plasmaprotease activity in septicemia is another factor that can lead to asplitting of the transferrin molecule (Esparza I., Brock JH. BiochemBiophys Acta 1980; 622:297-304; Doring GM. et al. Infect Immun 1988; 56:291- 293). The transferrin fragments that are released in this way andhave complexed iron can also catalyze oxygen radical formation byneutrophil granulocytes (Bradley EB., Edeker BL. J Clin Invest 1991; 88:1092-1102). The oxygen radicals can, on the one hand, cause directdamage to the cell membrane; on the other hand, they may damage theorganism indirectly by increasing prostaglandin synthesis.

SUMMARY OF THE INVENTION

It is the object of this invention to provide a therapeutic agentsuitable as a means of controlling the biological effects of endotoxinto a sufficient degree by virtue of its ability to neutralize largeamount of endotoxin and thus suitable for use as an efficient agent forprophylactic and therapeutic treatment of the toxic effects ofendotoxins. The agent should also be capable of minimizing theundesirable formation of harmful oxygen radicals in endotoxemia.

The invention thus provides for the use of apotransferrin that has bounddivalent zinc or copper ions. These apotransferrin-metal complexes arealso suitable for combination with immunoglobulin preparations oraddition to a plasma protein solution or a serum preserve.

DETAILED DESCRIPTION OF THE INVENTION

In vitro and in vivo experiments were carried out to test the capacityof the complexes of apotransferrin with divalent zinc or copper ions aswell as the combination of these complexes with immunoglobulins toinactivate endotoxins.

In vitro experiments:

A) Quantitative investigations of influence of transferrin-metalcomplexes on biological activity:

Apotransferrin-zinc and apotransferrin-copper as well as each of thesecombined with immunoglobulins were incubated with increasing amounts ofendotoxin at 37° C. for 60 minutes. Following incubation, quantitativedetermination of the biological activity of endotoxin in the supernatantwas carried out with a modification of the Limulus test using achromogenic substrate (Nitsche et al. In: Watson SW, Levin J, NovitskyTJ (eds.) DETECTION OF BACTERIAL ENDOTOXINS WITH THE LIMULUS AMEBOCYTELYSATE TEST, pp. 417-429 (1987)). The lower detection limit in thismodified version of the Limulus test is 1 EU/dl. The endotoxin activitymeasured by the Limulus test corresponds to the biological activity ofthe endotoxin (Nitsche D, Ulmer A., Flad HD: Endotoxin determination bythe Limulus test and the biological activity of endotoxin. Does acorrelation exist?--in preparation). Experiments carried out prior tothe endotoxin measurements did not reveal any influence on the Limulusreaction by Zn²⁺ or Cu²⁺ ions.

The apotransferrin used in the experiments was nearly free of metal ions(Atf.-0). No zinc was detected in this apotransferrin (Atf.-0) by meansof atomic absorption spectroscopy. No copper was found in the samples,either. The iron assay method described by Megraw and Bouda (Megraw RE,Hritz AM, Babson AL and Carroll JJ (1973) Clin. Biochem. 6: 266; Bouda J(1968) Clin. Chem. Acta 21:159) revealed no iron content in the 5%solution of apotransferrin. The lower detection limit of this method is0.3 μg/dl. Various amounts of zinc chloride (ZnCl₂) were added to theapotransferrin (Atf.-0) to obtain apotransferrin-zinc solutions with azinc content of 4.8 μg/g of apotransferrin (Atf.-Zn(A)) as well asapotransferrin with a zinc content of 598 μg/g of apotransferrin(Atf.-ZnB)).

Various amounts of copper(II) chloride (CuCl₂) were added to theapotransferrin (Atf.-0) to obtain apotransferrin-copper solutions with acopper content of 57 μg/g of apotransferrin (Atf.-Cu(A)) as well asapotransferrin-copper with a copper content of 740 μg/g ofapotransferrin (Atf.-Cu(B)).

Apotransferrin-iron was used as reference. Iron(III) chloride (FeCl₃)was added to apotransferrin (Atf.-0) so as to produce anapotransferrin-iron solution with an iron content of 598 μg of iron pergram of transferrin.

The following immunoglobulin preparations were used: 7S IgG(Sandoglobin^(R)) as well as 7S IgG enriched with 12% IgM(IgG/A/M=Pentaglobin^(R)).

Endotoxins from the following bacterial strains were used: Salmonellaabortus equi (NOVOPYREXAL^(R)), Pseudomonas aeruginosa Fisher type 7, aswell as the FDA endotoxin standard von E.coli O113:H10:KO.

DETERMINATION OF THE "MEAN PERCENTAGE OF INACTIVATION"

The percentage of endotoxin inactivated in each case was calculated fromthe difference between the initial added endotoxin concentration and theendotoxin activity measured following incubation with the protein. The"mean percentage of inactivation" for each protein concentration for theentire range of endotoxin concentrations tested (10 EU/dl-1000 EU/dl)was calculated on the basis of the values determined in this way for thedifferent endotoxin concentrations tested.

DETERMINATION OF ENDOTOXIN INACTIVATION CAPACITY

In order to quantify the capacity of apotransferrin-copper,apotransferrin-zinc and apotransferrin-iron as well as the combinationof apotransferrin-zinc and apotransferrin-copper with immunoglobulins toinactivate endotoxin, the "INACTIVATION CAPACITY" was determined. Toimprove measurement precision, this parameter was not determined atequivalence, but rather at endotoxin excess concentrations of 10 EU/dland 100 EU/dl. This parameter was calculated on the basis of the amountof endotoxin (EU/dl) that had to be added to each protein preparation toreach a concentration of free endotoxins of 10 EU/dl or 100 EU/dl in thesupernatant following incubation (60 minutes, 37° C.) with the proteinpreparation. This endotoxin concentration was calculated for eachpreparation (apotransferrin-zinc, apotransferrin-copper, immunoglobulinand the combination of apotransferrin-zinc and apotransferrin-copperwith immunoglobulin) on the basis of the relation between the amount ofendotoxin added and the endotoxin concentration measured in thesupernatant following incubation. The inactivation capacity wasdetermined by a series of measurements in which endotoxin in increasingconcentrations (10 EU/dl, 25 EU/dl, 50 EU/dl, 100 EU/dl, 200 EU/dl, 300EU/dl, 500 EU/dl, 750 EU/dl and 1,000 EU/dl) was incubated for 60minutes together with the various protein preparations. Theconcentration of apotransferrin-zinc or apotransferrin-copper in thereaction preparation was 312.5 mg/dl. Measurements carried out with thecombination of apotransferrin-zinc or apotransferrin-copper withimmunoglobulin, the concentration of each protein component in thereaction mixture was 312 mg/dl.

The mean endotoxin concentration that can be inactivated by each proteinpreparation [mg/dl] under the given conditions is the amount ofendotoxin determined in this way minus the given amount of excessendotoxin (10 EU/dl or 100 EU/dl). In relation to a proteinconcentration of 100 mg/dl, this amount of endotoxin is a gauge of the"mean inactivation capacity (EU/100 mg)" of each apotransferrin-metalcomplex or combination of an apotransferrin-metal complex withimmunoglobulin.

RESULTS

1.) APOTRANSFERRIN-METAL COMPLEX:

When endotoxin is incubated with apotransferrin free of metal ions(Atf.-0), an average reduction in endotoxin activity of 32.2% to 43.6%is registered, depending on the type and concentration of endotoxin.

When endotoxin is incubated with apotransferrin-zinc (Atf.-Zn(A)) with azinc content of 4.8 μg/g of apotransferrin, an average reduction inendotoxin activity of 72.5% to 82.4% is registered, depending on thetype and concentration of endotoxin.

When endotoxin is incubated with apotransferrin-zinc (Atf.-Zn(B)) with azinc content of 598 μg/g of apotransferrin, a reduction in endotoxinactivity of 91.4% to 97.8% is registered, depending on the type andconcentration of endotoxin.

When endotoxin is incubated with apotransferrin-copper (Atf-Cu(A)) witha copper content of 57 μg/g of apotransferrin, an average reduction inendotoxin activity of 84% to 89.3% is registered, depending on the typeand concentration of endotoxin.

When endotoxin is incubated with apotransferrin-copper (Atf.-Cu(B)) witha copper content of 740 μg/g of apotransferrin, a reduction in endotoxinactivity of 90.8% to 96.7% is registered, depending on the type andconcentration of endotoxin.

When endotoxin is incubated with apotransferrin-iron (Trf.-Fe) with aniron content of 598 μg/g of apotransferrin, an average reduction inendotoxin activity of 92.2% to 97.6% is registered, depending on thetype of endotoxin and the amount added.

a) On the basis of an endotoxin excess of 10 EU/dl, the inactivationcapacity is:

I.) Atf.-0: For S.abortus equi 1.8 EU/100 mg, for E.coli 2.3 EU/100 mgand for Pseudomonas aeruginosa 2.7 EU/100 mg.

II.) Atf.-Zn(A): For S.abortus equi 11.1 EU/100 mg, for E. coli 16.3EU/100 mg and for Pseudomonas aeruginosa 14.9 EU/100 mg.

III.) Atf.-Zn(B): For S.abortus equi 66.8 EU/100 mg, for E. coli 87.02EU/100 mg and for Pseudomonas aeruginosa 72.49 EU/100 mg.

IV.) Atf.-Cu(A): For S.abortus equi 34.6 EU/100 mg, for E. coli 43.8EU/100 mg and for Pseudomonas aeruginosa 39.7 EU/100 mg.

V.) Atf.-Cu(B): For S.abortus equi 70.1 EU/100 mg, for E. coli 79.4EU/100 mg and for Pseudomonas aeruginosa 64.6 EU/100 mg.

VI.) Trf.-Fe: For S.abortus equi 76.7 EU/100 mg, for E. coli 78.2 EU/100mg and for Pseudomonas aeruginosa 73.8 EU/100 mg.

b) On the basis of an endotoxin excess of 100 EU/dl, the inactivationcapacity is:

I.) Atf.-0: For S.abortus equi 17.8 EU/100 mg, for E.coli 19.4 EU/100 mgand for Pseudomonas aeruginosa 20.6 EU/100 mg.

II.) Atf,-Zn(A): For S.abortus equi 109.7 EU/100 mg, for E.coli 137.4EU/100 mg and for Pseudomonas aeruginosa 145.5 EU/100 mg.

III.) Atf.-Zn(B): For S.abortus equi 507.6 EU/100 mg, for E. coli 578.5EU/100 mg and for Pseudomonas aeruginosa 647.3 EU/100 mg.

IV.) Atf.-Cu(A): For S.abortus equi 362 EU/100 mg, for E. coli 387.4EU/100 mg and for Pseudomonas aeruginosa 409 EU/100 mg.

V.) Atf.-Cu(B): For S.abortus equi 522.4 EU/100 mg, for E. coli 527EU/100 mg and for Pseudomonas aeruginosa 585.1 EU/100 mg.

VI.) Trf.-Fe: For S.abortus equi 418.7 EU/100 mg, for E. coli 571.1EU/100 mg and for Pseudomonas aeruginosa 643.9 EU/100 mg.

2.) APOTRANSFERRIN-METAL COMPLEX and 7S IgG: The combination ofapotransferrin-zinc or apotransferrin-copper with a 7S IgG preparationimproves inactivation of endotoxin. Incubation of endotoxin withapotransferrin (312 mg/dl) and 7S IgG reduces endotoxin activity by amaximum of 61% to 68% at endotoxin concentrations under 100 EU/dl whenapotransferrin (Atf.-0) is used that is free of zinc, copper and iron.In the endotoxin concentration range above 100 EU/dl, this combinationreduces endotoxin activity by only 47.1% to 54.9%.

When apotransferrin-zinc (Atf.-Zn(A)) with a zinc content of 4.8 μg ofzinc per gram of apotransferrin is used in combination with the 7S IgGpreparation, a reduction in endotoxin activity of 83.1% to 89.1% isregistered, depending on the concentration and the type of endotoxinused.

When apotransferrin-zinc (Atf.-Zn(B)) with a zinc content of 598 μg pergram of apotransferrin is used in combination with the 7S IgGpreparation, endotoxin activity is reduced by 94.4% to 97.8%, dependingon the concentration and the type of endotoxin.

When apotransferrin copper(Atf.-Cu(A)) with a copper content of 57 μgper gram of apotransferrin is used in combination with 7S IgG, endotoxinactivity is reduced by 87.2% to 93.1%, depending on the concentrationand the type of endotoxin.

a) On the basis of an endotoxin excess of 10 EU/dl, the inactivationcapacity is:

I.) Atf.-0 and 7S IgG: For S.abortus equi 6.1 EU/100 mg, for E.coli 10.6EU/100 mg and for Pseudomonas aeruginosa 11.4 EU/100 mg.

II.) Atf.-Zn(A) and 7S IgG: For S.abortus equi 24.2 EU/100 mg, for E.coli 30.1 EU/100 mg and for Pseudomonas aeruginosa 27.5 EU/100 mg.

III.) Atf.-Zn(B) and 7S IgG: For S.abortus equi 74.1 EU/100 mg, for E.coli 98.9 EU/100 mg and for Pseudomonas aeruginosa 86.8 EU/100 mg.

IV.) Atf.-Cu(A) and 7S IgG: For S.abortus equi 40.1 EU/100 mg, for E.coli 50.6 EU/100 mg and for Pseudomonas aeruginosa 46.3 EU/100 mg.

b) On the basis of an endotoxin excess of 100 EU/dl, the inactivationcapacity is:

I.) Atf.-0 and 7S IgG: For S.abortus equi 28.6 EU/100 mg, for E.coli30.7 EU/100 mg and for Pseudomonas aeruginosa 39.1 EU/100 mg.

II.) Atf.-Zn(A) and 7S IgG: For S.abortus equi 151.5 EU/100 mg, for E.coli 177.9 EU/100 mg and for Pseudomonas aeruginosa 195.5 EU/100 mg.

III.) Atf.-Zn(B) and 7S IgG: For S.abortus equi 538.4 EU/100 mg, for E.coli 602.7 EU/100 mg and for Pseudomonas aeruginosa 675.1 EU/100 mg.

IV.) Atf.-Cu(A) and 7S IgG: For S.abortus equi 554.7 EU/100 mg, for E.coli 547.3 EU/100 mg and for Pseudomonas aeruginosa 608.1 EU/100 mg.

3.) APOTRANSFERRIN-ZINC and IgG/A/M (12% IgM): The addition ofapotransferrin-zinc to an IgG preparation enriched with IgA and 12% IgM(IgG/A/M) causes a further improvement in inactivation capacity. Whenendotoxin is incubated with the combination of IgG/A/M (12% IgM) (312mg/dl) and apotransferrin-zinc (Atf,-Zn(A) (312 mg/dl) with a zinccontent of 4.8 μg is used, endotoxin activity is reduced by 84 7% to 926% depending on the concentration and the type of endotoxin.

When IgG/A/M (12% IgM) (312 mg/dl) is combined with apotransferrin-zinc(Atf.-Zn(B) (312 mg/dl) with a zing content of 598 μg, endotoxinactivity is reduced by 94.8 to 98.1%, depending on the concentration andthe type of endotoxin.

a) On the basis of an endotoxin excess of 10 EU/dl, the inactivationcapacity is:

I.) Atf.-Zn(A) and IgG/A/M: For S.abortus equi 26.9 EU/100 mg, forE.coli 31.5 EU/100 mg and for Pseudomonas aeruginosa 24.9 EU/100 mg.

II.) Atf.-Zn(B) and IgG/A/M: For S.abortus equi 93.8 EU/100 mg, for E.coli 108.5 EU/100 mg and for Pseudomonas aeruginosa 97.8 EU/100 mg.

b) On the basis of an endotoxin excess of 100 EU/dl, the inactivationcapacity is:

I.) Atf.-Zn(A) and IgG/A/M: For S.abortus equi 141.1 EU/100 mg, forE.coli 173.2 EU/100 mg and for Pseudomonas aeruginosa 192.5 EU/100 mg.

II.) Atf.-Zn(B) and IgG/A/M: For S.abortus equi 688 EU/100 mg, for E.coli 828 EU/100 mg and for Pseudomonas aeruginosa 796.9 EU/100 mg.

B) Influence on release of the mediators ILi and IL 6

The release of IL 1 and IL 6 from monocytes was used as an additionalparameter for the degree of inactivation of the biological activity ofthe endotoxins by the complexes of divalent zinc ions withapotransferrin. Endotoxin was incubated with apotransferrin-zinc or withthe combination of apotransferrin-zinc and immunoglobulin (60 min.,37°). The supernatant was then incubated with monocytes and theconcentration of the mediators interleukin 1 and interleUkin 6 releasedby the monocytes was measured in the supernatant. Apotransferrin-zinc(Atf.-Zn(A)) with a zinc content of 4.8 μg per gram of apotransferrin aswell as apotransferrin-zinc (Atf.-Zn(B)) with a zinc content of 598 μgper gram of apotransferrin were used.

The following immunoglobulins were used: 7S IgG (IgG/A/M) enriched with12% IgM (Pentaglobin).

Endotoxins from the following bacterial strains were used: Salmonellaabortus equi (NOVOPYREXAL^(R)) and the FDA endotoxin standard EC-5 fromE. coli 0113:H10:K0.

Apotransferrin-zinc and apotransferrin-zinc in combination withimmunoglobulin were incubated together with endotoxin at 37° C. for 60minutes. Heat-denatured 7S IgG (80°, 10 min.) with an identical zinccontent in each case (4.6 μg or 598 μg of zinc/g of 7S-IgG) was used asa reference protein. The concentration of proteins in the preparationswas 312 mg/dl respectively. Incubation was carried out with endotoxinconcentrations of from 50 EU/dl to 500 EU/dl.

Following incubation, each endotoxin solution was incubated withmononuclear cells from healthy donors (16 hours at 37° C.). Then theconcentration of the released mediators IL 1 and IL 6 was determined. IL1 was measured by means of the fibroblast proliferation test (Loppnow,H.; Flad, H.-D.; Ulmer, A.-J. et al. "Detection of Interleukin 1 withHuman Dermal Fibroblasts" Immunbiol., 1989, 179, 283-291) using anEL4-6.1 thymoma cell line. The Concentration of IL 6 was also determinedby means of a proliferation test using an IL 6-dependent murinehybridoma (7TD1) (Van Damme J. Cayphas S. et al. (1987) Eur. J. Biochem.168: 543-550; as well as Van Oers MHJ. Van der Heyden A. and Aarden LA.(1988) Clin. exp. Immunol. 71: 314-419).

RESULTS

The incubation of endotoxin with apotransferrin-zinc resulted in areduction in the endotoxin-induced release of mediators from themononuclear cells. This effect is dependent on the zinc load of theapotransferrin--it can be improved by raising the zinc content.Combining apotransferrin-zinc with immunoglobulins further intensifiesthe inhibitive effect of the apotransferrin-zinc complex on the releaseof mediators.

I.) REFERENCE PROTEIN (heat-inactivated 7S IgG)

Following incubation of endotoxin (500 EU/dl) with heat-inactivated 7SIgG to which zinc had been added, and which was used as the referenceprotein, no difference in the amount of mediators released wasdetermined between the reference protein with a zinc content of 4.6 μgper g (Zn(A)) and the reference protein with a zinc content of 598 μgper g (Zn(B)). IL-1: Following incubation of the reference protein withendotoxin from S. abortus equi, the mean release of IL-1 was 2,685 U/ml;following incubation with endotoxin from E. coli IL-1 release was 2,968U/ml. IL-6: Following incubation of the reference protein with endotoxinfrom S. abortus equi, the mean release of IL-6 was 3,621 U/ml; followingincubation with endotoxin from E. coli IL-6 release was 5,974 U/ml.

II.) APOTRANSFERRIN-ZINC

a) Following incubation of 500 EU/dl of endotoxin with Atf.-Zn(A) theaverage release of ILlwas 321 U/ml with S. abortus equi and 412 U/mlwith E. coli. Average release of IL6 following incubation withapotrans-ferrin-zinc (Atf.-Zn(A)) was 1,132 U/ml with endotoxin from S.abortus equi and 1,825 U/ml with E. coli endotoxin.

b) Following incubation of 500 EU/dl of endotoxin with Atf.-Znf(B) theaverage release of IL 1 was 102 U/ml with S. abortus equi and 47 U/mlwith E. coli. Average release of IL6 following incubation withapotrans-ferrin-zinc (Atf.-Zn(B)) was 526 U/ml with endotoxin from S.abortus equi and 316 U/ml with E. coli endotoxin.

III.) APOTRANSFEPaIN-ZINC and IgG/A/M (12% IgM)

The combination of apotransferrin-zinc with a 7S IgG preparationenriched with IgA and 12% IgM (IgG/A/M) had the following results at animmunoglobulin concentration of 312 mg/dl--and an apotransferrin-zincconcentration of 312 mg/dl as well--after incubation with 500 EU/dl ofendotoxin:

a) With the combination of IgG/A/M with Atf.-Zn(A) the average releaseof IL 1 was 227 U/ml with endotoxin from S. abortus equi and 267 U/mlwith endotoxin from E. coli. Release of IL 6 following incubation was872 U/ml with endotoxin from S. abortus equi and 631 U/ml with E. coliendotoxin.

b) With the combination of IgG/A/M (12%) with Atf.-Zn(B) the averagerelease of IL 1 was 31 U/ml with endotoxin from S. abortus equi and 17U/ml with endotoxin from E. coli. Average release of IL6 followingincubation under these conditions was 186 U/ml with endotoxin from S.abortus equi and 137 U/ml with E. coli endotoxin.

C) Influence of apotransferrin complexes with zinc or copper ions onplasma endotoxin activity

Human plasma from healthy donors was diluted 1:10 and inactivated byheating (80° C., 10 min.). Apotransferrin-zinc or apotransferrin-copperwas then added. The concentration of apotransferrin-zinc in the plasmasample was 280 mg/dl and that of apotransferrin-copper 284 mg/dl. Thesame amount of albumin-zinc or albumin-copper was added to the referenceplasma samples with the same zinc or copper concentration in each case.Endotoxin (Pseudomonas endotoxin and E. coli endotoxin) was then addedto the plasma to which apotransferrin-zinc or apotransferrin-copper andalbumin-zinc or albumin-copper had been added, to reach an endotoxinactivity level of 300 EU/dl or 1,000 EU/dl in the plasma sample, whichwas then incubated for 60 minutes at 37° C. Following incubation, theendotoxin activity in the sample was measured using the Limulus test.

Plasma endotoxin activity was seen to be significantly reduced in thesamples by addition of apotransferrin-zinc or apotransferrin-copper ascompared to those to which only albumin (albumin-zinc or albumin-copper)was added.

a) Following incubation of endotoxin with albumin-zinc, plasma endotoxinactivity was a maximum of approx. 4% lower than the activity level ofthe added endotoxin, at a zinc content of 4.8 μg/g of albumin (Zn(A)) aswell as at a zinc content of 598 μg/g of albumin (Zn(B)). A nearlyequivalent endotoxin activity reduction, approx. 4%, was recordedfollowing incubation with albumin-copper with a copper content of 740 μgof copper per gram of albumin.

b) The addition of Atf,-Zn(A) to the plasma resulted in a reduction ofendotoxin activity in plasma by 83.4%±4.2% (n=20) at an addedconcentration of 300 EU/dl of endotoxin and a reduction by 69.1% ±6.8%(n=18) at an added concentration of 1,000 EU/dl of endotoxin.

c) The addition of apotransferrin-zinc Atf.-Zn(B) with a zinc content of598 μg of zinc per gram of apotransferrin to the plasma resulted in areduction of plasma endotoxin activity following incubation by93.9%±2.9% (n=20) at an added concentration of 500 EU/dl of endotoxinand a reduction by 94.8%±2.7% (n=20) at an added concentration of 1,000EU/dl of endotoxin.

d) The addition of apotransferrin-copper Atf.-Cu(B) with a coppercontent of 740 μg of copper per gram of apotransferrin to the plasmaresulted in a reduction of plasma endotoxin activity followingincubation by 92.7% ±4.1% (n=16) at an added concentration of 500 EU/dlof endotoxin and a reduction by 90.8%±3.4% (n=16) at an addedconcentration of 1,000 EU/dl of endotoxin.

Summary Evaluation of Results

1.) Apotransferrin complexes with divalent zinc or copper ions arecapable of reducing the biological activity of endotoxin, whereby thelevel of effectiveness depends on the apotransferrin-complexconcentration used, and are therefore well-suited as an agent forprophylactic and therapeutic treatment of the toxic effects ofendotoxins in the organism.

2.) Apotransferrin that is free of metal ions has only a slight effecton endotoxin activity.

3.) The degree to which apotransferrin-zinc or apotransferrin-copperreduces the toxic effects of endotoxin increases with the amount of zincor copper bound by the apotransferrin. The zinc or copper content of theapotransferrin used should be at least 0.1 μg per gram ofapotransferrin.

4.) The use of apotransferrin complexed with divalent zinc or copperions instead of trivalent iron ions as an agent for treatment of thetoxic effects of endotoxins in the organism results in endotoxinneutralization capacity nearly equivalent to that of transferrin andoffers the great advantage that administration of theseapotransferrin-metal complexes does not stimulate the formation of toxicoxygen are in the organism.

5.) The zinc ions added along with the apotransferrin-zinc complex aswell as the copper ions in the apotransferrin-copper are capable ofactivating the enzyme "superoxide-dismutase" as well as a number ofperoxidases. Thus the administration of apotransferrin-zinc orapotransferrin-copper has the decisive advantage over other prophylacticand therapeutic treatments of the toxic effects of endotoxins in theorganism that, in addition to the neutralization of endotoxins, animproved elimination of the toxic oxygen radicals produced in largeamounts in endotoxemia is achieved as well.

6.) Use of apotransferrin-zinc for endotoxin neutralization also has theadvantage of not only achieving endotoxin neutralization, but ofstimulating protein synthesis and thus, potentially, wound healingprocesses as well, since zinc ions facilitate DNA and RNA synthesis.

7.) The apotransferrin-zinc or apotransferrin-copper complexes can becombined with plasma protein solutions, whereby the immunoglobulinsolutions may contain either IgG alone or the other plasma proteins aswell, in particular IgM and IgA.

8.) The combination of apotransferrin-zinc and apotransferrin-copperwith other plasma proteins, in particular with immunoglobulins of theIgG and IgM fractions, achieves a significantly greater reduction of thebiological activity of endotoxins than does each individual componentalone.

9.) The synergistic effect in neutralizing endotoxins makes thecombination of apotransferrin-zinc and apotransferrin-copperparticularly well-suited for therapeutic measures to reduce the toxiceffects of endotoxin in all diseases in which an increased and prolongedtransfer of endotoxins into the bloodstream is to be expected.

The various aspects of the invention are further described by thefollowing examples:

EXAMPLE 1

The following experiments were carried out with apotransferrin-zinc toobtain an objective measure of the significance of the divalent zinc orcopper ion load of apotransferrin for inactivation of endotoxin inplasma:

Human plasma from healthy donors was diluted 1:10 with 0.9% NaCl, theninactivated by heat (80° C., 5 minutes). Apotransferrin complexed withvarying amounts of zinc was then added to the plasma. The apotransferrinconcentration in these reaction preparations was 300 mg/dl. The zincload of the apotransferrin was adjusted by adding varying amounts ofZnCl₂ to obtain apotransferrin-to-zinc mol ratios of 28:1, 1:1 and 1:3.

After the addition of apotransferrin-zinc to the inactivated plasma,endotoxin from E. coli in varying concentrations was also added to theplasma samples, resulting in endotoxin concentrations in the reactionpreparation of 50, 100, 200, 300, 500, 750, 1,000 and 1,500 EU/dl. Thepreparation was incubated for 60 minutes at 37° C. immediately after theaddition of endotoxin. Residual endotoxin activity in the samples wasthen measured. The inactivation capacity of the variousapotransferrin-zinc solutions for an endotoxin excess of 10 EU/dl wasthen calculated on the basis of the values obtained in this way.

The inactivation capacity values for 100 mg/dl of apotransferrin-zincwere calculated as a function of apotransferrin-to-zinc mol ratios. Thecalculations were carried out so as to arrive at an endotoxin excess of10 EU/dl:

a) 28:1:19.2 EU per 100 mg of apotransferrin-zinc

b) 1:1:87.1 EU per 100 mg of apotransferrin-zinc

c) 1:3:279.8 EU per 100 mg of apotransferrin-zinc

Inactivation capacity can be increased by raising the proportion of zincin the apotransferrin solution. For example, raising the zinc level froman apotransferrin-to-zinc mol ratio of 28:1 to 1:3 in the apotransferrinpreparation increases inactivation capacity by a factor of 14.5. It isexpected that, at a mol ratio of 1:3, the majority of apotransferrinmolecules will have bound two molecules of zinc each.

EXAMPLE 2

To simulate the conditions occurring in therapy of gram-negativeinfections with antibiotics, an endotoxin increase in the blood wasinduced in animal experiments by first inoculating bacteria into theperitoneal cavity, then administering a bactericidal antibioticintravenously, thus causing rapid bacterial decay. A blood collectioncatheter was inserted into the right jugular vein of anesthetized Wistarrats (250-300 g). A defined number of different species of bacteria wasthen inoculated into the animals' peritoneal cavity (E. coli, Klebsiellaspecies and Pseudomonas aeruginosa, each in a concentration of 7.5×10⁵cfu per kg of b.w., i.e. a total of 2.3×10⁶ cfu per kg of b.w.). 30minutes after the bacteria were inoculated into the abdominal cavity thepreparation under study (apotransferrin-zinc (Atf.-Zn(B) orapotransferrin-zinc in combination with immunoglobulin) was administeredi.v. through the venous catheter by means of a perfusor in a dosage of250 mg/kg b.w. The animals in the control group received a correspondingdosage of albumin-zinc in the same concentration and with the same zinccontent i.v. In order to induce an endotoxemia, a bactericidalantibiotic (IMIPENEM=Zienam^(R)) was administered i.v. one hour afterbacterial challenge. Blood samples for purposes of determining theendotoxin activity and the bacterial count in the blood were takenbefore bacterial inoculation as well as afterwards at 60-minuteintervals over a total period of 6 hours following bacterial challenge.

The following preparations were administered i.v.: apotransferrin(Atf.-Zn(B)) with a zinc content of 598 μg of Zn²⁺ /g, andapotransferrin-zinc (Atf.-Zn(B)) combined with IgM (12%)-enriched 7S IgG(IgG/A/M).

RESULTS

a) When albumin-zinc was administered i.v., plasma endotoxin activityincreased considerably following administration of the antibiotic. Anaverage endotoxin activity of 234 EU/dl was reached as early as one hourafter administration of the antibiotic. Five hours after antibioticadministration, mean plasma endotoxin activity had reached 278±31 EU/dl.

b) When apotransferrin-zinc (Atf.-Zn(B)) was administered i.v., theinitial increase in plasma endotoxin activity--one hour afteradministration of the antibiotic--was reduced by approx. 68.3%±7.2% incomparison to the albumin control group. At the end of the experiment,i.e. five hours after administration of the antibiotic, endotoxinactivity in the animals in the (Atf.-Zn(B)) group was still 63.5%±6.5%lower than in the albumin control group.

c) When apotransferrin (Atf.-Zn(A)) was administered i.v. in combinationwith an immunoglobulin (IgG/A/M) enriched with 12% IgM the initialincrease in plasma endotoxin activity--one hour after administration ofthe antibiotic--was reduced by approx. 73% in comparison to the albumincontrol group. Five hours after administration of the antibiotic,endotoxin activity in the animals that received this combination wasstill approx. 83.2%±7.9% lower than in the albumin control group.

The enhancement of inactivation capacity obtained with the combinationof apotransferrin with immunoglobulins makes it possible to control adrastic increase in plasma endotoxin activity, even in cases whereendotoxin continues to enter the bloodstream over a long period.Application of apotransferrin-zinc (Atf.-Zn(B)) alone results in areduction of endotoxin activity, in the initial phase only, similar tothat resulting from the apotransferrin-zinc/immunoglobulin combination.Unless either the dosage or the zinc load is raised, the inactivationcapacity of apotransferrin-zinc alone will not suffice in cases ofprolonged endotoxin passage into the blood, which may in turn lead to aresurgence of plasma endotoxin activity. The combination ofimmunoglobulin with apotransferrin-zinc represents a very efficienttherapeutic alternative, even when apotransferrin-zinc with a zinccontent under 500 μg/g of apotransferrin is used.

I claim:
 1. Method of reducing blood endotoxin activity in a patientcontaining endotoxins comprising intravenously administering atherapeutically effective amount to reduce blood endotoxin activityemploying apotransferrin bound to divalent zinc or copper cationsforming an apotransferrin-zinc or apotransferrin-copper complex.